Dawn of the organoid era

Novel and updated approaches of culturing cells in 3D are rapidly advancing our understanding of development, health, and disease. As tissues have been found to behave more realistically in 3D than in 2D cultures, organoid technology in combination with recent advances in the isolation and generation of stem cells, has rapidly become a promising concept in developmental and regenerative research. The development of all kinds of tissues can now be studied “in a dish,” allowing more detailed observations of stem cell maintenance, morphogens, and differentiation. This review explores how organoids have revolutionized academic research over the last 4 decades, and how they may continue to do so. It also addresses remaining hurdles in 3D cell culturing, and how they may be overcome.     

A microfluidic chemostat for experiments with bacterial and yeast cells

Bacteria and yeast frequently exist as populations capable of reaching extremely high cell densities. With conventional culturing techniques, however, cell proliferation and ultimate density are limited by depletion of nutrients and accumulation of metabolites in the medium. Here we describe design and operation of microfabricated elastomer chips, in which chemostatic conditions are maintained for bacterial and yeast colonies growing in an array of shallow microscopic chambers. Walls of the chambers are impassable for the cells, but allow diffusion of chemicals. Thus, the chemical contents of the chambers are maintained virtually identical to those of the nearby channels with continuous flowthrough of a dynamically defined medium. We demonstrate growth of cell cultures to densely packed ensembles that proceeds exponentially in a temperature-dependent fashion, and we use the devices to monitor colony growth from a single cell and to analyze the cell response to an exogenously added autoinducer.     

The Effects of Mycoplasma Contamination upon the Ability to Form Bioengineered 3D Kidney Cysts

Mycoplasma contamination of cell cultures is a pervasive, often undiagnosed and ignored problem in many laboratories that can result in reduced cell proliferation and changes in gene expression. Unless contamination is specifically suspected, it is often undetected in two dimensional (2D) cultures and the resulting effects of mycoplasma contamination are rarely appreciated and can lead to incorrect conclusions. Three dimensional (3D) tissue cultures are increasingly utilized to explore tissue development and phenotype. However, 3D cultures are more complex than 2D cell cultures and require a more controlled cellular environment in order to generate structures necessary to mimic in vivo responses and are often maintained for longer time periods. Changes to the microenvironment are assumed to have a more extreme effect upon the success of 3D tissue cultures than 2D cell cultures, but the effects of mycoplasma have not been studied. To test this hypothesis, we grew 2D cell cultures and 3D tissues from pig kidney epithelial cells (LLC-PK1) that were contaminated with mycoplasma and the same stock of cells after mycoplasma removal. We did not observe an effect of mycoplasma contamination on proliferation in 2D monolayer cell culture. However, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed. These data serve to reinforce the necessity of testing cell stocks for mycoplasma contamination.     

Significance of the clonal and sporadic chromosome abnormalities in non-neoplastic renal tissue

Summary Trisomy 7, trisomy 10 and loss of the Y chromosome have been found by some authors in presumptive normal parts of human kidneys. We describe cytogenetic findings in short-term cultures from 58 biopsies obtained from non-neoplastic and neoplastic (renal cell carcinoma, RCC) tissues from the same kidney, the same types of tissues from independent kidneys, and tissue from kidneys without neoplasia. The results indicate the following. Non-neoplastic tissue from kidneys involved in RCC have (in mosaics) trisomies 5, 7, 10, 18 and loss of the Y as non-random clonal changes. They are not the result of local metastasis but are also found in kidneys with non-tumoral chronic pathologies and should thus not be considered specific for RCC. They are neither culturing artefacts nor a general phenomenon found in cultured normal solid tissues, but are acquired abnormalities, possibily related to various reactive cellular states in the tissues that are histologically normal.     

Human peripheral blood T lymphocyte proliferation after activation of the T cell receptor: effects of unsaturated fatty acids

Oils enriched in certain polyunsaturated fatty acids suppress joint pain and swelling in rheumatoid arthritis patients with active synovitis. Because T lymphocyte activation is important for propagation of joint tissue injury in patients with rheumatoid arthritis, we examined the effects of fatty acids added in vitro on proliferation of human T lymphocytes stimulated with monoclonal antibodies to CD3 and CD4. Unsaturated fatty acids reduced T cell proliferation in a dose dependent manner (dihomogammalinolenic acid > gammalinolenic acid > eicosapentaenoic acid > arachidonic acid). Removal of fatty acids from cultures before cell stimulation did not change the effects, but addition of fatty acids after cell stimulation failed to reduce T cell responses. The saturated palmitic acid did not influence T cell growth. These studies indicate that small changes in cellular fatty acids can have profound effects on early events in T cell signaling and on T cell function.     

Contact-dependent regulation of growth of diploid human fibroblasts is dependent upon the presence of terminal galactose residues on plasma membrane glycoproteins

The growth of diploid human fibroblasts has previously been shown to be regulated mainly by the extent of cell-cell contacts [R. J. Wieser and F. Oesch (1986) J. Cell Biol. 103, 361], these contacts being effective only when terminal, beta-glycosidically linked galactose residues were present on plasma membrane glycoproteins. These studies, in which a high cell density in sparse cell cultures has been mimicked by the addition of immobilized plasma membrane glycoproteins, have been further extended to investigate the role of terminal galactose residues directly in cell cultures. The studies presented herein show that (i) culturing human fibroblasts in the presence of beta-galactosidase resulted in an approximately twofold higher saturation density, as well as a twofold higher proliferation rate at high cell densities when compared to the rates found in control cultures. (ii) The presence of alpha-lactalbumin in the culture medium, which acts as a modifier of the activity of galactosyltransferase, had the same effect as beta-galactosidase. (iii) Addition of the lectin I from Bandeiraea simplicifolia (BS I), which is specific for terminal galactose residues, resulted in an increase in the proliferation rate of cell cultures at high cell densities, while the proliferation was not affected at low cell densities. These data show that the presence of terminal, beta-glycosidically linked galactose is vital for the efficient growth control of normal cells.     

The taxonomy of obligate anaerobic bacteria

Studies on the chemotaxonomy of obligate anaerobic bacteria have been made. The combination of gas chromatography and mass spectrometry with computer-assisted analysis, permitting the multicomponent analysis of all products of bacterial metabolism and bacterial cell components, has been shown to be a research method, quite suitable for such studies. The chromatographic profiles of the end products of metabolism in anaerobic cultures of different age have been found to differ not in the set and number of peaks indicating various metabolites, but only in the concentration of metabolites, increasing in the process of prolonged incubation. The authors believe that the national microbiological "library" of the chromatographic profiles of anaerobic organisms should be created and the album of typing chromatographic profiles should be published; besides, data on new profiles should regularly appear in magazines.     

Cell cultures derived from embryos and melanoma of poeciliid fish

In poeciliid fish, melanoma of different degrees of malignancy can be produced by crossing specific genotypes. For a detailed investigation of the processes leading to proliferation or differentiation of the melanoma cells, it is necessary to establish cell cultures. The aim of the present study was to find out the optimal conditions for initiating and culturing poeciliid fish cells for the purpose of establishing cell cultures of melanoma. The optimal method was developed by using small pieces of late embryos as starting material and includes: (a) dispersion of tissue by mild stepwise treatment with a trypsin-EDTA mixture at low temperature; (b) culture of cells in the complex medium 199; (c) supplementation of medium with high percentage (20%) of fetal bovine serum; and (d) stabilization of pH by buffering the medium with HEPES. Under these conditions, primary and secondary cultures of embryonic cells have been initiated. An epithelial-like cell line has been subcultured for more than 80 passages. The method developed for embryonic tissues was used to start cell cultures from melanoma of platyfish-swordtail hybrids. Until now, only cells of rapidly growing malignant albino melanoma could be maintained in primary cultures. Secondary cultures could not be initiated since the melanoma cells tended to differentiate and stopped growing before a confluent monolayer was formed.     

The appropriateness of swab cultures for the release of human allograft tissue

Surgeries utilizing human allograft tissues have increased dramatically in recent years. With this increase has come a greater reliance on the use of swab culturing to assess allograft tissues for microbial contamination prior to distribution. In contrast to the typical industrial microbiological uses for swabs, the tissue banking industry has relied on swab cultures as a sterility release method for allograft tissues. It has been reported in the literature that swabs have limitations, both in sensitivity and reproducibility, so their suitability as a final sterility release method was evaluated in this study. Two different swab-culturing systems were evaluated (COPAN, EZ Culturette) using human allograft tissues spiked with low levels of multiple bacterial and fungal microorganisms. The average microbial recoveries for all challenge microorganisms for each tissue type and each swab system were calculated. Percent recoveries for each challenge microorganism were also calculated and reported. The results indicated that both swab systems exhibited low and highly variable recoveries from the seeded allograft tissues. Further analysis indicated there was no statistical difference (∝=0.05) between the two swab systems. It is the recommendation of the authors that swab culturing not be used to assess relatively low levels of microbial contamination on allografts. Instead, alternative validated microbial detection methods with improved sensitivity and reproducibility should be employed and validated for this critical task.     

Effect of simvastatin on the uptake and metabolic conversion of palmitic, dihomo-gamma-linoleic and alpha-linolenic acids in A549 cells

It is well known that simvastatin affects cholesterol synthesis. Furthermore it inhibits growth and proliferation and perturbs fatty acid metabolism in some cell lines. We have studied the effects of simvastatin on the uptake and metabolism of exogenous fatty acid in the human lung adenocarcinoma A549 cells. Simvastatin inhibited the proliferation of A549, and caused an increment in phospholipid/cholesterol ratio due to an increment in phospholipid content without affecting cholesterol content. All the fatty acids were uptaken and metabolized in both control and treated cells. The conversion of palmitic, linoleic and dihomo-gamma-linoleic acids to their metabolites and products/precursor ratios for the desaturation and elongation reactions showed that simvastatin enhanced the Delta5 desaturation step and altered some elongating steps. The machinery for unsaturated fatty acid synthesis in A549 is quite sensitive to simvastatin and its effects could have important implication taking into account that highly unsaturated fatty acids are involved in the regulation of diverse cellular functions by themselves or through their metabolites.     

Lipoxygenase inhibitors enhance the proliferation of human B cells

The addition of drugs which inhibit the lipoxygenase pathways of arachidonic acid metabolism to 5 day cultures of mitogen-stimulated human B cells enhanced the proliferative response more than 10-fold. Several chemically dissimilar lipoxygenase inhibitors increased proliferation in this system, whereas the specific cyclooxygenase inhibitor indomethacin had no effect. A lipoxygenase inhibitor could be added as late as 48 to 72 h after the initiation of culture and still cause a significant increase in B cell proliferation. These drugs increased the proliferation of both peripheral blood B cells and tonsillar B cells activated by Staphylococcus aureus Cowan I or anti-Ig M antibodies, in combination with a crude T cell supernate, a commercial B cell growth factor preparation, or recombinant lymphotoxin. A similar effect was observed in tonsillar B cells purified by counterflow centrifugal elutriation to remove esterase positive accessory cells, suggesting this is a direct effect on the B cell. Lipoxygenase blockade also caused a greater than twofold increase in polyclonal Ig production. The enhanced proliferation caused by lipoxygenase blockade could not be reversed by adding back exogenous leukotrienes or hydroxyeicosatetraenoic acids to the cultures. Furthermore, B cells prelabeled with [3H]arachidonic acid did not produce radiolabeled lipoxygenase metabolites of arachidonic acid under the same culture conditions in which the addition of lipoxygenase inhibitors had a profound effect on proliferation. Thus, lipoxygenase inhibitors markedly stimulate B cell proliferation under a variety of experimental conditions, although the mechanism responsible for this action has not yet been elucidated.     

Immunomodulatory effects of umbilical cord‐derived mesenchymal stem cells

Umbilical cord blood (UCB) is of great interest as a source of stem cells for use in cellular therapies. The immunomodulatory effect of mesenchymal stem cells (MSCs) originating from bone marrow, adipose tissue and amniotic membrane has previously been reported. In this study, MSCs were isolated from UCB with the aim of evaluating their immunomodulatory effects on proliferation of PB lymphocytes by two different techniques; namely, 5‐bromo‐2‐deoxyuridine ELISA and a carboxy fluorescein diacetate succinimidyl ester flow cytometric technique. MSCs were isolated from UCB, propagated until Passage four, and then characterized for cell surface markers by flow cytometry and ability to differentiate towards osteocytes and adipocytes. Immunosuppressive effects on PB lymphocytes were examined by co‐culturing mitomycin C‐treated UCB MSCs with mitogen‐stimulated lymphocytes for 72 hr. Thereafter, proliferation of lymphocytes was detected by CFSE flow cytometry and colorimetric ELISA. The titers of cytokines in cell culture supernatant were also assayed to clarify possible mechanisms of immunomodulation. UCB MSCs suppressed mitogen‐stimulated lymphocyte proliferation, which occurs via both cell‐cell contact and cytokine secretion. Titers of transforming growth factor beta and IL 10 increased, whereas that of IFN‐γ decreased in the supernatants of co‐cultures. Thus, UCB MSCs suppress the proliferation of mitogen‐stimulated lymphocytes. However further in vivo studies are required to fully evaluate the immunomodulatory effects of UCB MSCs.     

Conditioned media from solid tumor cell lines treated with retinoic acids both decreases and increases proliferation of capillary endothelial cells.

These studies investigated the effects of retinoic acids on endothelial cell proliferation. Three human neoplastic cell lines, U-373 MG glioblastoma, DU-145 prostate carcinoma, and TCCSUP bladder transitional cell carcinoma, were treated with all-trans, 9-cis, or 13-cis retinoic acids at 0.0001 to 10 microM. Hypoxia was used to ensure the expression of the angiogenic phenotype. Conditioned media (CM) were prepared by hypoxic culturing of the tumor cells with retinoic acids for 24 hours. Then CM were transferred to bovine capillary endothelial cells for 48 hours of normoxic culturing, counted and compared to controls. CM from U-373 MG and DU-145 cells, but not TCCSUP cells, treated with all-trans or 9-cis retinoic acids at several concentrations below 1 microM, caused significant (P<0.05) increases in endothelial cell proliferation of between 13 to 18%. Both nonconditioned and conditioned media, for retinoic acid concentrations above 1 microM, inhibited endothelial cell proliferation. All CM for 13-cis retinoic acid decreased endothelial cell proliferation. These results show that the cytotoxicity of retinoic acids and the growth promoting/inhibiting ability of the conditioned media is retinoic acid isoform, time, concentration, and cell type dependent. Most importantly, the conditioned media from tumor cells treated with low concentrations of all-trans or 9-cis retinoic acids significantly increased endothelial cell proliferation.     

Effect of catalase on the proliferation of human lymphocytes to phorbol myristate acetate

Phorbol esters have been documented to stimulate the proliferation of human blood mononuclear cell cultures. In addition, these agents are also known to stimulate the production and release of reactive oxygen species by monocytes. We demonstrated previously that H2O2, one of these oxygen metabolites, impairs the proliferative capacity of human blood lymphocytes. Therefore, in these experiments, we determined whether or not the H2O2 released by monocytes after activation by PMA modifies the proliferation of lymphocytes to this agent. Human blood mononuclear cells (80% lymphocytes and 20% monocytes) were incubated with PMA, and lymphoblastic transformation (LBT) was quantitated at 3 and 5 days by pulsing the cultures with thymidine. Initial experiments established that the concentration of PMA required for optimal LBT was 50 ng/ml. We then demonstrated that this concentration of PMA also induces a burst in hexose monophosphate shunt activity and H2O2 production of mononuclear cells as indicated by the enhanced oxidation of 14C-glucose and 14C-formate, respectively. The amount of H2O2 released into the medium was substantial. Our measurements indicate that the concentration of H2O2 could reach values as high as 0.008 mM during the first 2 hr of the cultures. The addition of catalase to PMA-treated cultures in concentrations sufficient to scavenge the H2O2 released by the monocytes was associated with an enhanced thymidine uptake (mean 79%). These results indicate that the hydrogen peroxide released by the monocytes modifies the response of lymphocytes to the PMA. Paradoxically, mononuclear cell cultures depleted of monocytes also had a lower proliferation to PMA than mononuclear cell cultures. This observation indicates that monocytes also produce factors required for lymphocyte proliferation to PMA such as an interleukin. In contrast, to PMA cultures, catalase did not alter the proliferation of mononuclear cell cultures stimulated by PHA. We previously documented that PHA does not stimulate an immediate burst in the oxidative metabolism of mononuclear cultures. Therefore, the effect of catalase in these two culture systems appears to correlate with the capacity of the mitogen to stimulate the oxidative metabolism of mononuclear cells. These observations suggest that the release of reactive oxygen species by monocytes may modify the response of lymphocytes to antigens both in vitro and in vivo.     

High-Resolution Proton NMR Studies of Lymphocyte Extracts

Anatomic imaging is now a well-developed application of magnetic resonance. Greater capabilities for physiologic characterization should become possible by concomitant application of spectroscopic methods. High-resolution in vitro spectroscopy must first provide a framework upon which in vivo and diagnostic interpretation may be based. Biochemical profiles consisting of quantitation of extracted aqueous metabolites and lipids of particular cells or organs establish an in vitro glossary for what may be found in the intact cell or living subject. A large variety of amino acids, intermediary metabolites, membrane precursors, and nucleotides are detectable in extracts of human peripheral blood lymphocytes, and significant changes in intracellular concentrations have been monitored after lectin-induced activation. Corresponding changes in lipid profile have also been noted. An increasing variety of other cells and tissues are being similarly characterized. Despite its limitations, NMR analysis possesses the unique prospect of providing a noninvasive and nondestructive source of biochemical information.     

Cultivation of Marine Sponges

There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important secondary metabolites in this group of animals. In this article, different approaches to producing sponge biomass are reviewed, and several factors that possibly influence culture success are evaluated. In situ sponge aquacultures, based on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass in bulk. However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment. Hence, a better-defined production system would be desirable. Some progress has been made with culturing sponges in semicontrolled systems, but these still use unfiltered natural seawater. Cultivation of sponges under completely controlled conditions has remained a problem. When designing an in vitro cultivation method, it is important to determine both qualitatively and quantitatively the nutritional demands of the species that is to be cultured. An adequate supply of food seems to be the key to successful sponge culture. Recently, some progress has been made with sponge cell cultures. The advantage of cell cultures is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However, this technique is still in its infancy: a continuous cell line has yet to be established. Axenic cultures of sponge aggregates (primmorphs) may provide an alternative to cell culture. Some sponge metabolites are, in fact, produced by endosymbiotic bacteria or algae that live in the sponge tissue. Only a few of these endosymbionts have been cultivated so far. The biotechnology for the production of sponge metabolites needs further development. Research efforts should be continued to enable commercial exploitation of this valuable natural resource in the near future. Received November 5, 1998; accepted June 20, 1999.     

Variable lymphocyte responses in rats after space flight.

Most studies of human blood lymphocyte function following space flight have indicated that microgravity suppresses T cell proliferation. However, several other postflight experiments with animals have shown no decrease in proliferation of lymphocytes from peripheral lymphatic tissues, suggesting that different tissues may be variably affected by microgravity. Therefore, we examined the proliferation of lymphocytes from both spleen and lymph nodes of rats following a 4-day flight aboard the Space Shuttle. The experiments were designed to investigate tissue variability as well as potential mechanisms involved in suppressing proliferation. We found that proliferation of lymph node lymphocytes (LNL) from flight (FLT) animals stimulated with the antigen receptor-dependent T cell mitogen concanavalin A was depressed and could not be restored by supplementing cultures with interleukin 1 or interleukin 2 (IL-2). Response to another receptor-dependent mitogen, phytohemagglutinin, was not decreased. However, proliferation of FLT LNL following stimulation with the receptor-independent, mitogenic combination of phorbol ester and ionomycin was depressed. LNL IL-2 activity, cell surface marker expression, and B cell responses to mitogen were normal. Thus, deficits in antigen receptor/ligand interactions, cell surface marker expression, or IL-2 did not account for the suppressed lymphocyte proliferation observed postflight. In contrast to LNL, FLT splenocyte proliferation was not depressed. Assayable IL-2, IL-2 receptor expression, and cell surface marker expression likewise were unaffected by space flight. The differences between lymph node and splenic responses demonstrate the tissue-specific nature of microgravity effects on individual lymphatic tissues.     

Detection of conjugated dienoic fatty acids in human vascular smooth muscle cells treated with conjugated linoleic acid

Conjugated linoleic acids (CLA) have attracted scientific interest due to their potential beneficial effects on atherosclerosis. Recent studies demonstrated that conjugated metabolites of CLA are found in tissues of CLA-fed animals and cultured cells treated with CLA. This observation has gained in importance since it has recently been shown that these metabolites of CLA exert specific biological activities. Therefore, the present study aimed to explore the potential formation of metabolites of cis-9, trans-11 CLA, trans-10, cis-12 CLA and trans-9, trans-11 CLA in cells of the vascular wall, which has not yet been shown. Examination of fatty acid composition of total cell lipids using Ag+-HPLC, GC-FID and GC-MS analysis revealed a significant isomer-specific formation of conjugated metabolites of CLA such as CD16:2, CD20:2 and CD22:2 in human coronary artery smooth muscle cells treated with various CLA isomers. Different CD16:2/CLA ratios between various CLA isomers as observed in the present study indicate that fatty acid metabolism is differently affected by the configuration of the double bonds. In conclusion, the observation from the present study suggests that the effects of CLA in vascular cells might not only be mediated by CLA itself but also by its conjugated metabolites. Future studies using highly purified conjugated metabolites of CLA are necessary to study their role in mediating biological effects of CLA in cell culture systems.     

In vitro Decline in Plant Cultures: Detection of a Legion of Covert Bacteria as the Cause for Degeneration of Long-term Micropropagated Triploid Watermelon Cultures

In vitro cultures of triploid seedless watermelon cv. Arka Manik displayed a decline in shoot and root growth after 4–5 years of active culturing. Visibly clean cultures upon indexing on enriched media showed covert bacteria, and a significant improvement in proliferation and rooting in response to surface sterilization. The bacteria however survived endophytically. Low pH and reduced clarity of agar gelled medium were found to mask the expression of bacteria in the tissue culture medium. Gentamycin, streptomycin or broad-spectrum bactericide cefazolin provided as a 2 ml overlay in the medium in factorial combination at 0 or 50 mg l^–1 resulted in selective suppression of some bacteria depending on the treatment and eight bacterial clones comprising of four Gram-positive (Bacillus spp.) and four-Gram negative (3 × Pseudomonas spp. and 1 × Aeromonas sp.) strains were isolated from the cultures. Provision of 50 mg l^–1 gentamycin in 2 ml overlay in the multiplication or rooting medium coupled with occasional decontamination of cultures helped in circumventing the decline problem. The plants established in the field after 6 years of active in vitro culturing appeared normal and fertile suggesting the feasibility of keeping cultures for long periods, thus saving time and other resources. Freeing the cultures from covert bacteria was complicated by the presence of different bacterial types and this will be addressed later.